(a) Field of the Invention
The present invention relates to a cell line in which a potassium channel is exogenously expressed; more specifically, it relates to a transfected cell line prepared by introducing a vector containing a gene encoding a potassium channel into a cell line that stably expresses T-type α1H calcium channels.
On the other hand, the present invention relates to a high throughput screening method for inhibitors of T-type α1H calcium channels using the above cell line.
(b) Description of the Related Art
The T-type channel is a member of voltage-dependent Ca2+ channels activated by low voltage. Three subtypes (α1G, α1H and α1I) of the gene encoding the T-type channel have been found so far (Perez-Reyes et al., Nature, 391:896-900, 1998; Cribbs et al., Cir. Res., 83:103-109, 1998; Lee et al., J. Neurosci., 19:1912-1921, 1999). When the subtypes of T-type channel are expressed in heterologous expression systems, e.g., Xenopus oocytes and HEK293 cell lines, they show such biophysical characteristics as (a) activation of the channel at low voltage around −60 mV, (b) rapid activation or non-activation of the channel, (c) a remarkably slow deactivation and (d) small conductance.
According to the prior arts, the T-type α1H channel plays an important role in regulating neuronal excitability in the central nervous system (Huguenard et al., Annu. Rev. Physiol., 58:329-334, 1996). In addition, T-type α1H channel regulates a variety of biological functions such as the heartbeat (Hagiwara et al., J. Physiol., 395:233-253), hormone secretion (Cohen et al., Proc. Natl. Acad. Sci. USA, 85:2412-2416, 1988; Enyeart et al., Mol. Endocrinol., 7:1031-1040, 1993), contraction of smooth muscle (Akaike et al., J. Physiol., 416:141-160, 1989), reproduction (Arnoult et al., Proc. Natl. Acad. Sci. USA, 93:13004-13009, 1996) and cell growth and differentiation (Berridge, Neuron, 21:13-26, 1998; Guo et al., J. Mol. Cell Cardiol., 30:1095-1103, 1998; Kono et al., J. Cell Biol., 132:915-923, 1996), etc.
When the T-type α1H channel is over-expressed by genetic or other reasons, diseases like absence epilepsy (Huguenard et al., J. Neurosci., 14:5485-5502, 1994; Tsakiridou et al., J. Neurosci., 15:3110-3117, 1995; Kim et al., Neuron, 31:35-45, 2001), heart disease (ex, ventricular hypertrophy and hypertension) (Nuss et al., Cir. Res., 73:777-782, 1993; Martinez et al., J. Mol. Cell Cardiol., 31:1617-1625, 1999), neuropathic pain (Dogrul et al., Pain, 105:159-168, 2003) and prostatic cancer (Mariot et al., J. Biol. Chem., 277:10824-10833) might be developed.
Therefore, T-type α1H channel has been a major target of studies on new drug development, world-wide. The importance of the study on the mechanism regulating the characteristics of the T-type α1H channel (biophysical and pharmacological properties, gene expression and transport regulation, signal transduction, etc), in physiological and pathophysiological conditions, is in no doubt because such studies enable the discovery of candidates for a new drug.
In order to develop a new drug inhibiting the T-type α1H channel, it is important that organic chemists explore the lead chemicals and design and synthesize their related compounds. For a successful new drug development, a high throughput screening (HTS) system, which investigates whether or not the synthesized materials can inhibit the T-type α1H channel rapidly and effectively, has to be established first.
Whether the synthesized materials are T-type α1H channel inhibitors or not is confirmed by measuring the calcium current with the conventional electrophysiological (namely, patch-clamp) method. This method has an advantage of providing the most accurate information on ion channels but has a problem of limitation in data points (the number of test compounds) per unit time, so the method might be inadequate for detecting lots of materials in a short time.
Recently, a patch-clamp HTS system enabling the electrical measurement of the activity of a T-type α1H channel has been developed by Axon Instruments (USA). This system may be a possible choice for researchers in overcoming the above problem in the new drug development related to the T-type α1H channel. Still a question remains to be answered whether such system can satisfactorily process the sophisticated and complex sequence of the patch-clamp method.
An alternative to the electrophysiological HTS is a detection method for searching T-type α1H channel inhibitors by measuring fluorescence. Instead of measuring the calcium current, this method evaluates the activity of calcium channel indirectly by measuring the fluorescence intensity which increases in proportion to the calcium influx through T-type α1H channels using calcium-binding fluorescent dyes such as fura. However, this method has the disadvantage of not being able to regulate the opening and closing of T-type α1H channels, unlike such voltage clamp methods as the patch clamp.
Well aware of the above mentioned problems in conventional high throughput screening approaches, the inventors thus developed a cell line and a HTS method based on it. In the cell line, a potassium channel was introduced to HEK293 cells, which stably expresses T-type α1H channels. The cells activate T-type α1H channels when a high concentration of potassium chloride is added to the extracellular medium without any electrical stimulus through maintaining a high resting potential in them. From the experiments, it was confirmed that T-type α1H channels are activated by depolarization upon the addition of a high concentration of KCl outside the cell. We were able to detect the resultant changes in the Ca2+ influx with a high signal-to-noise fluorescence ratio. The present inventors thus completed this invention by confirming that the cell line of the present invention is the optimal cell line for the high throughput screening and broad-spectrum studies of T-type α1H calcium channel inhibitors.